Separation techniques such as blot-based techniques can be used to identify the presence of a particular target molecule in a sample. One blot-based technique, Western blotting, can be used to identify the presence of a particular protein in a sample through its interaction with an antibody specific for said protein. The proteins of a sample may be separated from each other by electrophoresis, transferred to a suitable membrane support, which is then interrogated with the specific antibody. The binding of antibody to the protein is visualised as a “spot” on the membrane, providing information as to its presence and location. The location may give information about the physical state of protein, for example glycosylation, phosphorylation or proteolysis.
A disadvantage of Western blotting and other immunological techniques is that the data generated are qualitative (unitless), which limits the information obtained from an experiment and does not provide quantitative results comprising units. Furthermore, considerable day-to-day variation in sensitivity is observed, which prevents ready comparison between experiments performed on separate occasions, particularly when the experiments are performed by different researchers. Accordingly, there is considerable inter-experimental variation which makes comparisons between experiments difficult and inaccurate. These shortcomings limit both the quality of information gained and the productivity of the technique.
There are a variety of other blot based detection techniques. Southern blotting is a technique used to detect the presence of a particular DNA sequence, whilst Northern blotting is used to locate a particular RNA sequence within a mixture. ELISA techniques are highly sensitive, and therefore able to detect very small amounts of protein or other antigenic substance in a sample. The basis of this method is the binding of the antigen by an antibody that is linked to a surface of a plate. Formation of an immune complex is detected by use of peroxidase coupled to an antibody, the peroxidase being used to generate an amplifying colour reaction. However, despite the highly sensitive nature of ELISA, it does not quantify the amount of protein or antigen present in the sample. Thus, the disadvantages described in connection with Western blotting are also relevant to other blot based detection techniques.
There are other separation based techniques such as High Performance Liquid Chromatography (HPLC) and isoelectric focusing. Isoelectric focusing techniques are techniques used to separate proteins which utilise differences in the isoelectric points of the proteins. The isoelectric point of a protein is the pH at which a protein has no net charge. Under those circumstances it will not migrate in an electric field. Isoelectric focusing techniques use a pH gradient set up between a cathode and an anode and proteins will migrate towards their isoelectric point. Isoelectric focusing techniques do not provide truly quantitative results.
It is therefore long been desired that simple, effectively reproducible calibration technology would correct the described shortcomings and provide quantitative data which are readily comparable between experiments.